Combination containing growth factors and polyelectrolytes

ABSTRACT

The invention relates to active substance combinations which consist of at least one polypeptide with the biological action of fibroblast growth factors and of at least one cationic polyelectrolyte. These combinations permit improved dosage of the FGF activity.

This is a continuation of the application Ser. No. 08/330,619, filedOct. 28, 1994, now U.S. Pat. No. 5,885,960, which in turn is a CONT ofSer. No. 07/950,566 filed Sep. 25. 1992, abandoned.

SUMMARY OF THE INVENTION

The invention relates to compositions which, besides at least onepolypeptide with the biological activity of fibroblast growth factors(FGF), contain at least one cationic polyelectrolyte.

Fibroblast growth factors (FGF) which belong to the class of endogenouspeptide growth factors, were originally detected as substances in thebrain and pituitary and isolated therefrom and showed an activitypromoting the growth of fibroblasts. FGFs are known to be effectiveangiogenic factors which are responsible, inter alia, forneovascularization in wound healing. More details on FGFs, includingtheir modification products, on their isolation and preparation, theirstructure, their biological activities and their mechanisms, and oncorresponding medical uses can be found in the specialist literaturewhich is now wide-ranging. A comprehensive review is provided, forexample, by A. Baird and P. Bohlen, Fibroblast Growth Factors in:Peptide Growth Factors and their Receptors I (editors: M. B. Sporn andA. B. Roberts) Springer Verlag Berlin, Heidelberg, New York 1990.

Growth factors regarded as suitable according to the invention are notonly the "classical" FGFs such as acidic fibroblast growth factor (aFGF)and basic fibroblast growth factor (bFGF) but also all peptides whichessentially display the biological activity of FGF.

FGFs in the narrower sense include natural FGFs especially of bovine andhuman origin, as well as FGFs prepared recombinantly. Particularlypreferred are human aFGF and bFGF prepared recombinantly. Details onbovine and human and aGFG and bFGF as prepared recombinantly can befound in the following patent documents, for example: EP 0 228 449, EP 0248 819, EP 0 259 953, EP 0 275 204. FGFs in the wider sense alsoinclude muteins which differ from aFGF and bFGF to a certain extent inthe number and/or sequence of the amino acids without thereby beingassociated with a substantial alteration in the action. In addition, thegeneric term is to be understood to cover, for example, various forms ofbFGF which differ in their length: they contain 146, 153, 154 or 157amino-acid residues. Finally, FGFs in the wider sense also embracerelated peptides, some of which have distinctly different amino-acidsequences but have the biological activity of FGF. The following patentdocuments may be mentioned by way of example with reference to theliterature: EP 0 148 922, EP 0 226 181, EP 0 281 822, EP 0 288 307, EP 0319 052, EP 0 326 907 and WO 89-12645. The said peptides are comprisedby "peptides with FGF activity" for simplification.

FGFs within the meaning of the invention furthermore include derivativesof these peptides which are obtained with stabilizing and/oractivity-increasing agents. These are, in particular, forms of aFGF andbFGF stabilized against acid, which contain as stabilizing agents, forexample, glycosaminoglycans such as heparin, heparin fragments, heparinsulfate and dermatan sulfate or glucan sulfates such as dextran sulfateand cyclodextrin sulfate. FGF derivatives of this type are described,for example, in EP 251 806, EP 267 015, EP 312 208, EP 345 660, EP 406856, EP 408 146, WO 89-12464, WO 90-01941 and WO 90-03797.

Preferred for the present invention are FGFs of human origin and themuteins thereof, especially bFGF. It is particularly preferred to usehuman bFGF prepared recombinantly, as described in EP 0 248 819.

It is common to the peptides with FGF activity that they bindspecifically to the FGF receptors of the cell membrane and then displaytheir biological action, for example in wound healing. The result ofthis binding is that, for example, bFGF cannot normally be detected inserum even after injection. For example, bFGF as a basic protein(IP=9.8) is bound to anionic macromolecules (for example, nucleic acidsand acidic components of the extracellular matrix (ECM) such as heparinsulfate--see, inter alia, Moscatelli et al.: Interaction of basicfibroblast growth factor with extracellular matrix and receptors.Lecture at a conference on "The Fibroblast Growth Factor Family"arranged by The New York Academy of Sciences, 1991). This property isdescribed in the literature and is utilized, inter alia, for theisolation of FGFs: purification of a- and bFGF by chromatography onsupports which contain bound heparin.

The non-specific binding of the peptides with FGF activity reduces theamount of FGF able to reach the specific receptor on the cell. In theparticular case of relatively old (chronic) wounds or burn wounds, orwhen large amounts of necrotic material are present, the non-specificbinding will absorb a large and, at the same time, unknown portion ofthe FGF. This is why accurate dosage of the effective amount of peptideswith FGF activity is impossible. Overdosage of the active substance isnecessary. This means that the dosage is also unreliable because it isunknown how many non-specific binding sites are present in theparticular wound.

Attempts to stabilize bFGF, to reduce its non-specific binding and toincrease its affinity for the specific receptor have hitherto entailedcombining bFGF with heparin or other sulfated glycans (for example,dextran sulfate) or sucralfate. However, at present, bFGF is normallytested without such additives. The only ancillary substances added arefor stability and improved administration.

The disadvantage of the use of pure bFGF and, to varying extents, alsothe said combinations and derivatives comprises the necessity foroverdosage of the active substance and the unreliability of dosagebecause it is unknown how many non-specific binding sites are present inthe particular wound.

An object of the invention is therefore to provide a combination ofactive substances whose biological activity is unaffected by thenon-specific binding of the peptides with FGF activity.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

It is true that EP 0 312 208 proposes employing gel formers togetherwith growth factors, preferably epidermal growth factor (EGF), forimproved dosage by means of diffusion control. However, this measuredoes not solve the fundamental problem of the dosage of growth factors,which results from the variable non-specific binding.

It has now been found that these dosage problems can be solved byadditional administration of at least one cationic polyelectrolyte onadministration of peptides with FGF activity. It is surprising in thisconnection that although the cationic polyelectrolytes block thenon-specific binding sites, for example in the ECM, nevertheless thepeptides with FGF activity are able to reach their specific receptors inthe cell membrane, and thus the biological activity of FGF is retained.The composition of this invention excludes the compositions of EP 0 312208 which incidently contain cationic polyelectrolytes, whose disclosureis hereby incorporated by reference.

The cationic polyelectrolyte typically is a polymer having a positivenet charge in solution and which lacks specific growth activity.

It is possible to use, according to the invention, a number of knowncationic polyelectrolytes. It is common to them that they irreversiblyform complexes under physiological conditions with cell structures orbinding sites which bind FGF non-specifically. Examples of suitablecationic polyelectrolytes are polyamines and -imines, as well as theirquaternized derivatives. Also suitable are polyguanidines, as well aspolypeptides which contain basic amino acids; for example, polylysine,polyornithine, polyarginine. It is also possible for polypeptides ofthese types to contain mixtures of a plurality of amino acids. The aminoacids themselves can be employed either in racemic form or in pure D orL form in the peptides used according to the invention. Also suitableare polysaccharides with basic groups. It is particularly preferred touse chitosans and their derivatives, for example, N-carboxybutylchitosanor methylpyrrolidonechitosan, because these are biodegradable and thedegradation products are identical to components of normal metabolism(glucosamine and N-acetylglucosamine). Cationic polyelectrolytes havingmolecule weights of 500 daltons or greater are preferred. It is alsopreferred that the cationic polyelectrolyte does not substantiallyincrease the solution viscosity, e.g., the value is, 2 cps or less,preferably is 1 cps or less and preferably is not increased at all.

It is possible according to the invention to employ the cationicpolyelectrolytes as free bases or else as salts with physiologicallytolerated organic or inorganic acids. Examples of physiologicallytolerated organic acids are glutamic acid, acidic acid and lactic acid.Hydrochloric acid is mentioned as example of a physiologically toleratedinorganic acid. Other physiologically tolerated acids are known to thepharmaceutical technologist.

These substances are administered in excess compared with the peptideswith FGF activity and at the same time as the latter. It is likewisepossible to administer the cationic polyelectrolytes first. This resultsin FGFs being able to diffuse unhindered to the specific receptor andbind thereto. However, it is also conceivable within the scope of theprocedure according to the invention to administer the cationicpolyelectrolytes subsequently in order to displace the FGFs from thenon-specific binding sites. The advantage of this procedure is thepossibility in each case of using a comparatively low dose of thepeptides with FGF activity, which results in a corresponding increase inthe therapeutic index. In addition, considerably more reliable use canbe achieved, since the FGFs are able to reach the target site even inthe case of highly necrotic wounds. Multiple administration is oftenindicated in such cases.

The invention therefore relates to combinations which contain at leastone polypeptide with FGF activity and at least one cationicpolyelectrolyte lacking specific growth activity.

The combinations according to the invention can in this connectionpreferably be in the form of aqueous solutions which, besides at leastone polypeptide with FGF activity and at least one cationicpolyelectrolyte, contain customary additives such as buffer substancesor salts to regulate the osmotic pressure or else fillers. Theconcentration of the polypeptides with FGF action in these solutions is0.1 ng/ml-500 μg/ml, preferably 0.5 ng/ml-300 μg/ml, and that of thecationic polyelectrolytes is 1 μg/ml-300 mg/ml, preferably 0.1 mg/ml-200mg/ml. These solutions can be further processed in a known manner byadding gel formers to give hydrogels. The solutions, where appropriatewith added fillers, or hydrogels can be dried or lyophilized to give webmaterials, to give films, to give powders, to give granules or to givethreads. Threads obtained in this way can be further processed to givewoven fabrics or nets. It is also possible to absorb these solutions inwound gauze or in wound closure film and to dry the latter. The driedpresentations can also be used, reconstituted with liquid, as aqueoussolution or as gel or hydrogel for wound dressing. Finally, thecombinations according to the invention can also be in the form of areagent assemblage (kit) in which the components (peptide with FGFactivity and cationic polyelectrolyte) are present separately but inbalanced amounts.

In the case of fresh, clean wounds, a small excess of cationicpolyelectrolyte, i.e., at least about five times the amount of peptideswith FGF activity, is sufficient for the masking. However, this excessmust be considerably increased appropriate for the state of the wound inorder to block the non-specific binding sites. For this reason it ispossible according to the invention for the amount of cationicpolyelectrolyte to be up to ten thousand times the amount of thepeptides with FGF activity. The ratio between the amounts by weight ofcationic polyelectrolyte lacking specific growth activity and peptidewith FGF activity is preferably 100-2,000.

The invention furthermore -relates to the use of an addition of at leastone cationic polyelectrolyte lacking specific growth activity to apreparation which contains at least one peptide with FGF activity inorder to block non-specific binding sites for the peptide with FGFactivity.

The invention relates to processes for the preparation of combinationswhich contain at least one peptide with FGF activity and, in addition,at least one cationic polyelectrolyte, wherein the components arepreferably present as aqueous solution, and furthermore can containcustomary additives such as buffer substances or salts to regulate theosmotic pressure or else fillers. These solutions can also besterilized. It is additionally possible to process these solutionsfurther to give semisolid or solid presentations, for example hydrogels,webs, threads, woven fabrics, granules, powders, impregnated materials.

The invention relates to processes for the preparation of pharmaceuticalcompositions, in which a combination which contains at least one peptidewith FGF activity and, in addition, at least one cationicpolyelectrolyte is converted with at least one solid, liquid orsemiliquid vehicle or ancillary substance into a suitable dosage form.

The invention relates to pharmaceutical compositions which containcombinations composed of at least one peptide with FGF activity and, inaddition, at least one cationic polyelectrolyte.

The invention relates to the use of combinations which contain at leastone peptide with FGF activity and, in addition, at least one cationicpolyelectrolyte for controlling diseases.

The invention relates to the use of a combination which contains atleast one peptide with FGF activity and, in addition, at least onecationic polyelectrolyte for the preparation of a pharmaceutical.

The combinations according to the invention can advantageously beemployed for the therapy of wounds for which FGF therapy is indicated,especially when free granulation processes are involved in the healingof these wounds or defects. Important areas of application of thecombinations according to the invention are ulcers of the skin (forexample pressure sores, diabetic gangrene, arterial and venouscongestion), in addition wounds from burns, mucosal ulcers and lesions,skin donation sites, skin and soft-tissue transplantation sites(preparation of the floor of the wound) and surgical wounds (especiallywhere healing functions are impaired). For all these areas ofapplication the administration of combinations of peptides with FGFactivity and suitable polycations is particularly important when thepatients' wound-healing capacity is impaired (for example because ofage, basic diseases or therapeutic measures).

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and unless otherwise indicated, allparts and percentages are by weight.

The entire disclosures of all applications, patents and publications,cited above and below, and of corresponding application German P 41 32005.0 filed Sep. 26, 1991, are hereby incorporated by reference.

EXAMPLES

The preparation and use of the combinations according to the inventionis described by way of example hereinafter, but this description is notto be regarded as a restriction of the subject matter of the invention.The advantages compared with the FGF-containing preparations known todate from the state of the art become clear from the reported examplesof use.

Example 1 Example 1a Chitosan Solution

100 mg of chitosan glutamate (manufactured by Protan Co., Norway) aredissolved in 10 ml of Ringer's solution.

Example 1b bFGF Solution

100 μg of human bFGF prepared recombinantly are dissolved in 10 ml of 20mM citrate buffer pH 5.0.

Example 1c Combined Chitosan-bFGF Solution

10 ml each of the solutions from Examples 1a and 1b are mixed. The finalconcentration of bFGF is 5 μg/ml.

Example 2 Example 2a Chitosan Solution

100 mg of methylpyrrolidone-chitosan (produced according to Muzzarelli,WO 91/02 168) are dissolved in 10 ml of aqueous sodium chloride solution(9 g/l).

Example 2b bFGF Solution

50 μg of human bFGF prepared recombinantly are dissolved in 10 ml of 20mM citrate buffer pH 5.0.

Example 2c Combined Chitosan-bFGF Solution

10 ml each of the solutions from Examples 2a and 2b are mixed. The finalconcentration of bFGF is 2.5 μg/ml.

EXAMPLE 3

After preparation of the solution from Example 1c it is lyophilized in alayer thickness of 2-5 mm. The result is a web which contains activesubstance and is suitable for covering wounds.

EXAMPLE 4

Commercially available wound dressing gauze is impregnated with asolution from Example 2c (2 ml/5 cm²) and then dried. The result is agauze which contains active substance and is suitable for coveringwounds.

Use Example A

The viscous solution from Example 1c is uniformly applied at about 1ml/5 cm² area of wound to the wound to be treated.

Use Example B

The wound is first pretreated with a solution from Example 1a (chitosan)about 1 ml/5 cm² area of wound). After a pretreatment time of about 30minutes, the excess solution and wound discharge are removed with aswab. The wound is subsequently treated further with a solution fromExample 1b (bFGF) (about 1 ml/5 cm² area of wound).

Use Example C

The wound is first pretreated with a solution from Example 1a (chitosan)(about 1 ml/5 cm² area of wound). After a pretreatment time of about 30minutes, the excess solution and wound discharge are removed with aswab. The wound is subsequently treated further with a solution fromExample 1c (combination of chitosan and bFGF) (about 1 ml/5 cm² area ofwound).

The latter method has the advantage that the application of thecombination of bFGF and chitosan results in bFGF-binding substanceswhich have been newly discharged into the area of the wound areabsorbed.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A pharmaceutical composition, consistingessentially of 0.1 ng/ml-500 μg/ml of an FGF peptide and a cationicpolyelectrolyte, wherein, when the composition is in solution, thecationic polyelectrolyte is bioavailable in an amount effective todecrease non-specific binding of the FGF peptide.
 2. A compositionaccording to claim 1, wherein the amount of cationic polyelectrolyte is1 μg/ml-300 mg/ml.
 3. A composition according to claim 2, wherein theamount of cationic polyelectrolyte is 0.1 mg/ml-200 mg/ml.
 4. Apharmaceutical composition of claim 1, wherein the cationicpolyelectrolyte is not chitosan.
 5. The pharmaceutical composition ofclaim 1, wherein the cationic polyelectrolyte is a chitosan derivative.6. A pharmaceutical composition of claim 1 further comprising apharmaceutically acceptable carrier.
 7. A pharmaceutical composition,consisting essentially of 0.1 ng/ml-500 μg/ml of FGF peptide and acationic polyelectrolytewherein the cationic polyelectrolyte isbioavailable in an amount effective to decrease non-specific binding ofthe FGF peptide, and wherein, when the composition is in solution, thecationic polyelectrolyte does not increase the viscosity of thesolution.
 8. A pharmaceutical composition of claim 7 further comprisinga pharmaceutically acceptable carrier.
 9. A pharmaceutical compositionof claim 1, wherein said cationic polyelectrolyte lacks specific growthactivity.
 10. A pharmaceutical composition of claim 7, wherein saidcationic polyelectrolyte lacks specific growth activity.
 11. Apharmaceutical composition of claim 1, wherein the weight ratio of thecationic polyelectrolyte to the FGF peptide is 5:1 to 10,000:1.
 12. Apharmaceutical composition of claim 1, wherein the weight ratio of thecationic polyelectrolyte to the FGF peptide is 100:1 to 2,000:1.
 13. Aweb material, comprising a dried pharmaceutical composition, consistingessentially of 0.1 ng/ml-500 μg/ml of an FGF peptide and a cationicpolyelectrolyte, wherein, when the composition is in solution, thecationic polyelectrolyte is bioavailable in an amount effective todecrease non-specific binding of the FGF peptide.
 14. A wound dressinggauze, consisting essentially of a dried pharmaceutical composition,comprising 0.1 ng/ml-500 μg/ml of an FGF peptide and a cationicpolyelectrolyte, wherein, when the composition is in solution, thecationic polyelectrolyte is bioavailable in an amount effective todecrease non-specific binding of the FGF peptide.